1. Glycolysis and Gluconeogenesis Alice Skoumalová

2. Metabolism of glucose - overview

4. 1. Glycolysis

5. Glucose: the universal fuel for human cells Sources:  diet (the major sugar in our diet)  internal glycogen stores  blood (glucose homeostasis) Glucose oxidation:  after a meal: almost all tissues  during fasting: brain, erythrocytes

6. Glycolysis:  oxidation and cleavage of glucose  ATP generation (with and without oxygen)  all cells  in the cytosol (the reducing equivalents are transferred to the electron-transport chain by the shuttle) ATP is generated: 1. via substrate-level phosphorylation 2. from NADH 3. from oxidation of pyruvate Regulation of glycolysis: 1. Hexokinase 2. Phosphofructokinase 3. Pyruvate Kinase Generation of precursors for biosynthesis:  fatty acids  amino acids  ribosis-5-P

7. Anaerobic glycolysis  a limited supply of O 2  no mitochondria  increased demands for ATP Lactic acidemia  in hypoxia

8. Phosphorylation of glucose:  irreversible Glucose 6-P:  cannot be transported back across the plasma membrane  a precursor for many pathways that uses glucose Hexokinases Glucokinase (liver, β-cell of the pancreas)  high K m

9. Michaelis-Menten kinetics

10. 1. Conversion of glucose 6-P to the triose phosphates 2. Oxidation and substrate-level phosphorylation

11. 1. Conversion of glucose 6-P to the triose phosphates irreversible regulation essential for the subsequent cleavage

12. Substrate-level phosphorylation 2. Oxidation and substrate-level phosphorylation

13. Summary of the glycolytic pathway: Glucosis + 2 NAD + + 2 P i + 2 ADP 2 pyruvate + 2 NADH + 4 H + + 2 ATP + 2 H 2 O ∆G 0´ = - 22 kcal (it cannot be reversed without the expenditure of energy!)

14. Clinical correlations: Hypoxemia (lack of oxygen in tissues)  Acute hemorrhage (hypotension, lost of erythrocytes) - anaerobic glycolysis - lactate formation, metabolic acidosis  Chronic obstructive pulmonary disease (an insuficient ventilation) - anaerobic glycolysis, lactate formation, metabolic acidosis - accumulation of CO 2, respiratory acidosis  Myocardial infarction (lack of oxygen in myocardium) - anaerobic glycolysis, lactate formation - lack of ATP

15. Aerobic glycolysis:  involving shuttles that transfer reducing equivalents across the mitochondrial membrane

16. Glycerol 3-phosphate shuttle:

17. Malate-aspartate shuttle:

18. Anaerobic glycolysis: Energy yield 2 mol of ATP dissociation and formation of H +

19. Daily lactate production115 (g/d) Erythrocytes29 Skin20 Brain17 Sceletal muscle16 Renal medulla15 Intestinal mucosa8 Other tissues10 Major tissues of lactate production: (in a resting state)

20. Cori cycle: Lactate can be further metabolized by:  heart, sceletal muscle Lactate dehydrogenase: a tetramer (subunits M and H)

21. Lactate dehydrogenase Pyruvate + NADH + H + lactate + NAD + LD 5 isoenzymes: Heart (lactate) Muscle (pyruvate)

22. Biosynthetic functions of glycolysis:

23. Clinical correlations: Long-intensity exercise (for example a sprint) - the need for ATP exceeds the capacity of the mitochondria for oxidative phosphorylation, anaerobic glycolysis → lactate formation, muscle fatigue and pain - a training → the amounts of mitochondria and myoglobin increase

24. Regulation

25. Fructose 2,6-bis-phosphate:  is not an intermediate of glycolysis!  Phosphofructokinase-2:inhibited through phosphorylation - cAMP-dependent protein kinase (inhibition of glycolysis during fasting-glucagon) tissue-specific isoenzymes (low K m, a high afinity) glucokinase (high K m ) the rate-limiting, allosteric enzyme tissue-specific isoenzymes

26. the liver isoenzyme - inhibition by cAMP-dependent protein kinase (inhibition of glycolysis during fasting) Lactic acidemia: increased NADH/NAD + ratioinhibition of pyruvate dehydrogenase

27. 2. Gluconeogenesis

28. Gluconeogenesis:  synthesis of glucose from noncarbohydrate precursors → to maintain blood glucose levels during fasting  liver, kidney  fasting, prolonged exercise, a high- protein diet, stress Specific pathways: 1.Pyruvate → Phosphoenolpyruvate 2.Fructose-1,6-P → Fructose-6-P 3.Glucose-6-P → Glucose

29. Precursors for gluconeogenesis 1.lactate (anaerobic glycolysis) 2.amino acids (muscle proteins) 3.glycerol (adipose tissue)

30. Conversion of pyruvate to phosphoenolpyruvate 1. Pyruvate → Oxaloacetate  Pyruvate carboxylase 2. Oxaloacetate → PEP  Phosphoenolpyruvate- carboxykinase

31. Conversion of phosphoenolpyruvate to glucose 3. Fructose-1,6-P → Fructose-6-P  Fructose 1,6-bisphosphatase (cytosol) 4. Glucose-6-P → Glucose  Glucose 6-phosphatase (ER)

32. Clinical correlations: Alcoholism - excessive ethanol consumption → increase NADH/NAD + ratio that drive the lactate dehydrogenase reaction toward lactate - lack of precursors for gluconeogenesis → its inhibition - insuficient diet - reduced glucose in the blood, consumption of glycogen in the liver → hypoglycemia

33. 3. Regulations

34. TypeMechanismExample Substrate concentrationSaturation kinetics (according Michaelis- Menten) Glucokinase (activation after meal- high K m ) AllostericConformational changes induced by the binding of the allosteric efector Enzymes of glycolysis and glukoneogenesis (allosteric effectors ATP, AMP, citrate) Covalent modificationConformational changes induced by phosphorylation by proteinkinases Phosphorylation og glycogensynthase and glycogenphosphorylase (glucagon) Protein-protein interaction Conformational changes as a result of different protein binding Muscle glycogenphosphorylase (activated by Ca 2+ -calmodulin) Proteolytic cleavageActivation by proteolytic cleavage of precursor molecule Proteins of coagulation cascade (zymogens) Enzyme synthesisInduction or repression of the enzyme synthesis Enzymes of gluconeogenesis (induced during fasting) Enzyme regulation:

35. Second messenger - cAMP: Glucagon Adenylate- cyclase

36. Regulation of gluconeogenesis:  concomitant inactivation of the glycolytic enzymes and activation of the enzymes of gluconeogenesis 1. Pyruvate → PEP Phosphoenolpyruvate carboxykinase - induced by glucagon, epinephrine, and cortisol 2. Fructose 1,6-P → Fructose 6-P Fructose 1,6-bisphosphatase - inhibited by fructose 2,6-P 3. Glucose 6-P → Glucose Glucose 6-phosphatase - induced during fasting

37. Summary Glycolysis Generation of ATP (with or without oxygen) The role of glycolysis in different tissues Lactate production Regulation (3 key enzymes) Gluconeogenesis Activation during fasting, prolonged exercise, after a high- protein diet Precursors: lactate, glycerol, amino acids 3 key reactions:Pyruvate → PEP Fructose-1,6-P→ Fructose-6-P Glucose-6-P → Glucose Regulation

39. Pictures used in the presentation: Marks´ Basic Medical Biochemistry, A Clinical Approach, third edition, 2009 (M. Lieberman, A.D. Marks)